Albiflorin Decreases Glutamate Release from Rat Cerebral Cortex Nerve Terminals (Synaptosomes) through Depressing P/Q-Type Calcium Channels and Protein Kinase A Activity

The purpose of this study was to investigate whether and how albiflorin, a natural monoterpene glycoside, affects the release of glutamate, one of the most important neurotransmitters involved in neurotoxicity, from cerebrocortical nerve terminals (synaptosomes) in rats. The results showed that albiflorin reduced 4-aminopyridine (4-AP)-elicited glutamate release from synaptosomes, which was abrogated in the absence of extracellular Ca2+ or in the presence of the vesicular glutamate transporter inhibitor or a P/Q-type Ca2+ channel inhibitor, indicating a mechanism of action involving Ca2+-dependent depression of vesicular exocytotic glutamate release. Albiflorin failed to alter the increase in the fluorescence intensity of 3,3-diethylthiacarbocyanine iodide (DiSC3(5)), a membrane-potential-sensitive dye. In addition, the suppression of protein kinase A (PKA) abolished the effect of albiflorin on glutamate release. Albiflorin also reduced the phosphorylation of PKA and synaptosomal-associated protein of 25 kDa (SNAP-25) and synapsin I at PKA-specific residues, which correlated with decreased available synaptic vesicles. The results of transmission electron microscopy (TEM) also observed that albiflorin reduces the release competence of synaptic vesicles evoked by 4-AP in synaptosomes. In conclusion, by studying synaptosomally released glutamate, we suggested that albiflorin reduces vesicular exocytotic glutamate release by decreasing extracellular Ca2+ entry via P/Q-type Ca2+ channels and reducing PKA-mediated synapsin I and SNAP-25 phosphorylation.


Introduction
The neurotransmitter glutamate has an important role in excitatory neurotransmission in the mammalian central nervous system (CNS), but excessive glutamate leads to excitotoxicity.This event is present in several neurological disorders, such as epilepsy, traumatic brain injury, stroke, and Alzheimer's disease [1].A high concentration of glutamate in the synaptic cleft leads to the overstimulation of glutamate receptors, which in turn leads to increased calcium levels, mitochondrial abnormalities, oxidative stress, and eventually cell atrophy and death [1].Therefore, a normal level of synaptic glutamate is necessary for the prevention of excitotoxicity, and this may potentially be achieved through the regulation of synaptic glutamate release [2].
Natural chemicals from food or herbs play a crucial role in the development of safe and effective treatments for CNS disease therapy.Albiflorin (C23H28O11, MW 480.5; Figure 1A), a monoterpene glycoside, is an active ingredient of the roots of Paeonia albiflora that is used in traditional Chinese medicine and has medicinal properties [3,4].Albiflorin has antioxidant, anti-inflammatory, immunoregulatory, analgesic, as well as neuroprotective effects, without causing overt toxicity [5][6][7][8].Regarding its neuroprotective effects, albiflorin has been demonstrated to reduce neuronal loss, attenuate brain damage, and alleviate mood and memory deficits in several experimental models of neurological disorders, including spinal cord injury, cerebral ischemia, depression, post-traumatic stress disorder, and Alzheimer's disease [9][10][11][12][13][14][15].However, to the best of our knowledge, no research has examined the effect of albiflorin on the regulation of synaptic glutamate release, which is a crucial mechanism for neuroprotective action [2,16,17].Therefore, the aim of this study was to examine the effect of albiflorin on the release of glutamate and the underlying mechanism using rat cerebral cortex nerve terminals (synaptosomes).Synaptosomes can accumulate, store, and release neurotransmitters and do not suffer from any postsynaptic interactions.Therefore, the preparation and modulation of synaptosomes is a well-established in vitro technique for investigating synaptic neurotransmitter release [18].
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 2 of 13 turn leads to increased calcium levels, mitochondrial abnormalities, oxidative stress, and eventually cell atrophy and death [1].Therefore, a normal level of synaptic glutamate is necessary for the prevention of excitotoxicity, and this may potentially be achieved through the regulation of synaptic glutamate release [2].Natural chemicals from food or herbs play a crucial role in the development of safe and effective treatments for CNS disease therapy.Albiflorin (C23H28O11, MW 480.5; Figure 1A), a monoterpene glycoside, is an active ingredient of the roots of Paeonia albiflora that is used in traditional Chinese medicine and has medicinal properties [3,4].Albiflorin has antioxidant, anti-inflammatory, immunoregulatory, analgesic, as well as neuroprotective effects, without causing overt toxicity [5][6][7][8].Regarding its neuroprotective effects, albiflorin has been demonstrated to reduce neuronal loss, attenuate brain damage, and alleviate mood and memory deficits in several experimental models of neurological disorders, including spinal cord injury, cerebral ischemia, depression, post-traumatic stress disorder, and Alzheimer's disease [9][10][11][12][13][14][15].However, to the best of our knowledge, no research has examined the effect of albiflorin on the regulation of synaptic glutamate release, which is a crucial mechanism for neuroprotective action [2,16,17].Therefore, the aim of this study was to examine the effect of albiflorin on the release of glutamate and the underlying mechanism using rat cerebral cortex nerve terminals (synaptosomes).Synaptosomes can accumulate, store, and release neurotransmitters and do not suffer from any postsynaptic interactions.Therefore, the preparation and modulation of synaptosomes is a well-established in vitro technique for investigating synaptic neurotransmitter release [18].

Reduced Glutamate Release from Albiflorin Is Mediated through Ca 2+ Channel Suppression
The ability of albiflorin to depress glutamate overflow from synaptosomes was further assessed.As shown in Figure 2, the release of glutamate elicited by 4-AP was greatly reduced by ω-conotoxin GVIA, an N-type Ca 2+ channel blocker [F(2,12) = 256.7,p < 0.0001], or ω-agatoxin IVA, a P/Q-type Ca 2+ channel blocker [F(2,12) = 233.3,p < 0.0001].In the presence of ω-conotoxin GVIA, albiflorin continued to significantly reduce the 4-APinduced release of glutamate (p < 0.0001).In contrast to the ω-conotoxin GVIA, the effect of albiflorin was prevented by the ω-agatoxin IVA pretreatment, and no significant difference was observed between the glutamate release after ω-agatoxin IVA treatment alone and after the ω-agatoxin IVA and albiflorin treatment (p = 0.8).

Albiflorin Failed to Affect the Synaptosomal Membrane Potential
The suppression of Ca 2+ channels by albiflorin might be due to an alteration in the synaptosomal plasma membrane potential, which consequently modulates Ca 2+ influx into the terminal.Therefore, the effect of albiflorin on the synaptosomal plasma membrane potential was assessed with the membrane-potential-sensitive dye DiSC 3 (5).As shown in Figure 3, 4-AP (1 mM) increased the fluorescence intensity of DiSC 3 (5).Compared with that of 4-AP alone, the presence of albiflorin did not significantly affect the increase in DiSC 3 (5)

Albiflorin Failed to Affect the Synaptosomal Membrane Potential
The suppression of Ca 2+ channels by albiflorin might be due to an alteration in the synaptosomal plasma membrane potential, which consequently modulates Ca 2+ influx into the terminal.Therefore, the effect of albiflorin on the synaptosomal plasma membrane potential was assessed with the membrane-potential-sensitive dye DiSC3 (5).As shown in Figure 3, 4-AP (1 mM) increased the fluorescence intensity of DiSC3 (5).Compared with that of 4-AP alone, the presence of albiflorin did not significantly affect the increase in DiSC3( 5

Albiflorin Failed to Affect the Synaptosomal Membrane Potential
The suppression of Ca 2+ channels by albiflorin might be due to an alteration in the synaptosomal plasma membrane potential, which consequently modulates Ca 2+ influx into the terminal.Therefore, the effect of albiflorin on the synaptosomal plasma membrane potential was assessed with the membrane-potential-sensitive dye DiSC3 (5).As shown in Figure 3, 4-AP (1 mM) increased the fluorescence intensity of DiSC3 (5).Compared with that of 4-AP alone, the presence of albiflorin did not significantly affect the increase in DiSC3( 5
To further explore the regulatory effect of albiflorin on presynaptic PKA pathway, we assessed the phosphorylation of SNAP-25 and synapsin 1, which are presynaptic substrates for PKA [19].PKA phosphorylates SNAP-25 on threonine 138 (T138) and synapsin I on serine 9 (S9), promoting the trafficking and mobilization of synaptic vesicles to nerve terminals and consequently increasing the size of the releasable vesicle pools [21,22].As shown in Figure 5A-E   To further explore the regulatory effect of albiflorin on presynaptic PKA pathway, we assessed the phosphorylation of SNAP-25 and synapsin 1, which are presynaptic substrates for PKA [19].PKA phosphorylates SNAP-25 on threonine 138 (T138) and synapsin I on serine 9 (S9), promoting the trafficking and mobilization of synaptic vesicles to nerve terminals and consequently increasing the size of the releasable vesicle pools [21,22].As shown in Figure 5A-E

Albiflorin Reduces the Release Competence of Synaptic Vesicles Evoked by 4-AP in Synaptosomes
The regulation of synaptic vesicles by albiflorin was further evaluated using transmission electron microscopy (TEM).As shown in Figure 6A,B, the synaptosome contained numerous synaptic vesicles (control group).Synaptosomes treated with 1 mM 4-AP showed significantly fewer synaptic vesicles than control synaptosomes [F(2,6) = 65.3,p < 0.001], indicating that 4-AP evokes the release of all released vesicles.However, the number of synaptic vesicles was greater in synaptosomes preincubated with 10 µM albiflorin for 10 min before 4-AP application than in those treated with 4-AP alone (p < 0.001).

Albiflorin Reduces the Release Competence of Synaptic Vesicles Evoked by 4-AP in Synaptosomes
The regulation of synaptic vesicles by albiflorin was further evaluated using transmission electron microscopy (TEM).As shown in Figure 6A,B, the synaptosome contained numerous synaptic vesicles (control group).Synaptosomes treated with 1 mM 4-AP showed significantly fewer synaptic vesicles than control synaptosomes [F(2,6) = 65.3,p < 0.001], indicating that 4-AP evokes the release of all released vesicles.However, the number of synaptic vesicles was greater in synaptosomes preincubated with 10 µM albiflorin for 10 min before 4-AP application than in those treated with 4-AP alone (p < 0.001).

Discussion
This work focused on evaluating the effects of albiflorin on the release of glutamate from synaptosomes acutely prepared from the cerebral cortex of adult rats.We report here that albiflorin reduced vesicular exocytotic glutamate release from cerebrocortical synaptosomes via the depression of Ca 2+ channel activation and the PKA pathway.

Discussion
This work focused on evaluating the effects of albiflorin on the release of glutamate from synaptosomes acutely prepared from the cerebral cortex of adult rats.We report here that albiflorin reduced vesicular exocytotic glutamate release from cerebrocortical synaptosomes via the depression of Ca 2+ channel activation and the PKA pathway.

The Mechanism by Which Albiflorin Inhibited the 4-AP-Evoked Glutamate Release
The K + channel blocker 4-AP, which mimics the physiological mechanisms of terminal depolarization and the Ca 2+ -dependent release of neurotransmitters, is considered a quasi-physiological stimulus for investigating the characteristics of the induced release of glutamate [23].In the present study, 1 mM 4-AP elicited the release of a large amount of glutamate from nerve terminals, consistent with previous studies [24,25].Albiflorin reduced this 4-AP-elicited glutamate release in a dose-dependent manner (3-30 µM) and reached a maximum effect at 18 µM.Furthermore, the albiflorin-mediated decrease in glutamate release was abolished by a blockade of vesicular glutamate transporters.This suggests that the albiflorin-mediated inhibition of 4-AP-elicited glutamate release is mediated by a decrease in the exocytotic pool available for release.The mechanisms possibly involved in the ability of albiflorin to inhibit vesicular exocytotic glutamate release were further assessed.In particular, the involvement of voltage-dependent Ca 2+ channels, which are primarily contributed to the activation of vesicle exocytosis, was investigated [26].Ca 2+ channels, which are primarily supported for glutamate exocytosis, are N-type and P/Q-type [19,27,28].Notably, we found that the depression of P/Q-type Ca 2+ channels was involved in the glutamate-release-inhibiting effect of albiflorin, as indicated by the blockage of the action of albiflorin by the P/Q-type Ca 2+ channel inhibitor, while the N-type Ca 2+ channel inhibitor was ineffective.The glutamate release measured in the presence of both ω-agatoxin IVA and albiflorin was not significantly different from that observed in the presence of ω-agatoxin IVA alone.The lack of additivity in the actions of albiflorin and ω-agatoxin IVA can be explained by the inhibition of the same release pathway by both compounds.Moreover, the effect of albiflorin on 4-AP-elicited glutamate release was also significantly affected in the absence of extracellular Ca 2+ .These results suggest that albiflorin depresses vesicular exocytotic glutamate release from synaptosomes by decreasing extracellular Ca 2+ entry via P/Q-type Ca 2+ channels.
Additionally, the inhibition of Na + channels or activation of K + channels causes presynaptic inhibition resulting from the hyperpolarization of nerve terminals.This results in a reduction in Ca 2+ influx and a consequent decrease in neurotransmitter release [29,30].This mechanism did not involve the inhibitory effect of 4-AP-elicited glutamate release by albiflorin observed in the present study.This notion is supported by our observation that albiflorin did not influence 4-AP-evoked plasma membrane depolarization in synaptosomes.Although no direct evidence has indicated that albiflorin acts on presynaptic Ca 2+ channels, our tentative conclusion is that the inhibitory effect of glutamate release by albiflorin occurs primarily through direct regulation of Ca 2+ channels to affect Ca 2+ entry.Further investigations are needed to better understand how albiflorin leads to the suppression of Ca 2+ channels.
In glutamatergic nerve terminals, PKA increases glutamate release, and the phosphorylation of synaptic proteins in the release machinery, such as SNAP-25 and synapsin I, could be involved [20].SNAP-25 and synapsin I are phosphorylated by PKA at T138 and S9, respectively.This phosphorylation promotes synaptic vesicle trafficking and increases the size of the releasable vesicle pools [22,31].In the present study, when albiflorin was combined with the PKA inhibitor H89, the inhibitory effect of 4-AP-elicited glutamate release observed was not different from that observed with H89 alone.The lack of synergy in the effects of albiflorin and H89 can be explained by the notion that both factors inhibit the same pathway.In addition, we also observed increased levels of p-PKA, p-138-SNAP-25, and p-S9-synapsin I in 4-AP-treated cortical synaptosomes, which may involve a 4-AP-induced increase in the number of releasable vesicles and therefore increasing glutamate exocytosis.Furthermore, we found that albiflorin significantly decreased the levels of p-PKA, p-T138-SNAP-25, and p-S9-synapsin I in 4-AP-treated cortical synaptosomes.Therefore, it seems that the suppression of PKA by albiflorin reduces the phosphorylation of synapsin I and SNAP-25, resulting in attenuation of the number of releasable synaptic vesicles and reduced glutamate release.Consistent with this hypothesis, TEM revealed that 4-AP-induced synaptic vesicle changes were significantly reduced in albiflorin-treated cortical synaptosomes.

Therapeutic Implications
Albiflorin has no obvious acute oral toxicity and is a safe natural drug ingredient [7].The ability of albiflorin to inhibit glutamate release from nerve terminals in the present study is of special interest, considering that excess glutamate is involved in many brain disorders, including neurodegenerative diseases (e.g., Alzheimer's disease and Parkinson's disease), and in mood disorders (e.g., depression) [32,33].The reduction in glutamate release by albiflorin needs further investigation, and this observation represents a crucial mechanism of action of albiflorin that might contribute to its therapeutic effect on brain disorders.We infer that the glutamate-release-inhibiting effects of albiflorin might contribute to the reported effects on Alzheimer's disease and depression [9,11,34].

Animals
Male Sprague Dawley rats weighing 180-200 g were purchased from the BioLASCO Taiwan Co., Ltd.(Taipei, Taiwan).The animal care and treatment were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.The experimental protocol was approved by the Fu Jen Catholic University Animal Care Committee (A11208).In brief, the animals were kept in a room with controlled environmental conditions, maintaining a steady temperature of 23 ± 3 • C, a relative humidity of 50 ± 10%, and a 12 h light/dark cycle.The animals were provided with a standard rodent diet and had unrestricted access to purified water.All efforts were made to minimize the number of animals used and to reduce their suffering.A total of 25 animals were utilized in the study.

Preparation of Synaptosomes
Synaptosomes were isolated using Percoll density gradient centrifugation [24].The experimental rats were sacrificed by decapitation.Their brains were quickly extracted and placed in a chilled medium containing 320 mM sucrose with a pH of 7.4.The cerebral cortex was then dissected and homogenized in the sucrose medium.The homogenate was centrifuged at 3000× g for 2 min at 4 • C. The resulting supernatant was then centrifuged again at 14,500× g for 12 min at 4 • C. The pellet was resuspended in a HEPES-buffered medium.The suspension was then separated using a layered, discontinuous Percoll gradient, which contained 320 mM sucrose, 1 mM EDTA, 0.25 mM DL-dithiothreitol, and Percoll concentrations of 3%, 10%, and 23%.The gradients were then centrifuged at 32,500× g for 7 min at 4 • C. Synaptosomes were recovered from the 10 and 23% Percoll bands and diluted in a HEPES buffer medium (HBM).This medium contained 140 mM NaCl, 5 mM KCl, 5 mM NaHCO 3 , 1 mM MgCl 2 •6H 2 O, 1.2 mM Na 2 HPO 4 , 10 mM glucose, and 10 mM HEPES, with a pH of 7.4.Following further centrifugation at 27,000× g for 10 min at 4 • C, the synaptosomal pellets were resuspended in HBM, and the protein content was determined by the Bradford assay (Thermo Fisher Scientific, Waltham, MA, USA).A volume of 0.5 mg of synaptosomal suspension was diluted in HBM and centrifugated (3000× g at 4 • C) for 10 min.The synaptosomal pellet was divided into three independent fractions used for the following purposes: (a) analysis of the glutamate release, plasma membrane potential, and Na + concentration; (b) evaluation of the expression of p-PKA, p-T138-SNAP-25, and p-S9-synapsin I by Western blot; and (c) evaluation of the changes in synaptic vesicles by TEM.

Glutamate Release Assay
The release of glutamate from purified cerebrocortical synaptosomes was monitored in real time using an assay.Exogenous glutamate dehydrogenase (GDH) and NADP + were used in this assay to couple the oxidative deamination of the released glutamate to the generation of NADPH.This process was detected using fluorometric methods [35,36].Synaptosomal pellets were resuspended in HBM containing 16 M bovine serum albumin to bind any free fatty acids.A 2 mL aliquot was transferred to a stirred, thermostatted cuvette at 37 • C and incubated in the presence of 1 mM NADP + , 50 units of glutamate dehydrogenase, and 1.2 mM CaCl 2 .The released glutamate undergoes oxidative deamination by GDH to produce NADPH, resulting in an increase in fluorescence.This increase in fluorescence, with excitation and emission wavelengths of 340 and 460 nm, respectively, was measured to monitor glutamate release.The fluorescence of NADPH was detected using LS-55B (PerkinElmer, Waltham, MA, USA) model spectrofluorimeter.At the end of each experiment, 5 nmol of exogenous glutamate was added as a standard.Data were obtained at 2 s intervals.The fluorescence response used to quantify the released glutamate after a 5 min depolarization with 1 mM 4-AP was expressed as nanomoles of glutamate per milligram of synaptosomal protein per 5 min (nmol/mg/5 min).

Plasma Membrane Potential
The plasma membrane potential was evaluated using DiSC3(5), a dye that is advantageous due to its ability to alter fluorescence intensity in response to changes in membrane potential [37].Synaptosomes were preincubated and resuspended following the protocol used for the glutamate release experiments.After 3 min of incubation, 5 µM DiSC3(5) was added and allowed to equilibrate.Following 4 min of incubation, CaCl 2 (1.2 mM) was introduced.At the 10 min mark, 4-AP (1 mM) was added to depolarize the synaptosomes.The fluorescence of DiSC3(5) was then monitored at excitation and emission wavelengths of 646 nm and 674 nm, respectively.The results were expressed in fluorescence units.

TEM
For ultrastructural analysis, purified cortical synaptosomes were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde for 12 h at 4 • C. The fixed cortical synaptosomes were rinsed in phosphate-buffered saline (PBS), then post-fixed in 1% osmium tetroxide for 2 h.Afterward, they were dehydrated and embedded in epoxy resin.Next, ultrathin sections were cut to a thickness of 70 nm using an ultramicrotome (EM UC7, Leica Microsystems, Wetzlar, Germany).A transmission electron microscope (JEM-1400, JEOL, Tokyo, Japan) was used to examine the ultrastructure of the cortical synaptosomes.

Data Analysis
The Shapiro-Wilk test was used to test the data normality.Data analysis and graph creation were performed using GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA).The results were analyzed using one-way analysis of variance (ANOVA), followed by Tukey's post hoc test when comparing more than two groups.A two-tailed Student's t-test was used to determine whether there was a significant difference between the means of two groups.Data were presented as mean ± standard error of the mean (SEM).The statistical significance was set at p < 0.05.

Conclusions
Our data demonstrated that by decreasing Ca 2+ entry via P/Q-type Ca 2+ channels and reducing PKA-mediated synapsin I and SNAP-25 phosphorylation, albiflorin inhibits vesicular exocytotic glutamate release from rat cerebral cortical synaptosomes.This investigation enhances the understanding of albiflorin action in the brain and suggests that albiflorin is valuable for treating glutamate-induced neurological disorders.

Figure 3 .
Figure 3. Albiflorin does not affect the synaptosomal membrane potential.The membrane potential of synaptosomes was measured using DiSC3(5) during depolarization with 4-AP (1 mM), either

Figure 3 .
Figure 3. Albiflorin does not affect the synaptosomal membrane potential.The membrane potential of synaptosomes was measured using DiSC3(5) during depolarization with 4-AP (1 mM), either

Figure 3 .
Figure 3. Albiflorin does not affect the synaptosomal membrane potential.The membrane potential of synaptosomes was measured using DiSC3(5) during depolarization with 4-AP (1 mM), either without (control) or with albiflorin, which was added 10 min prior to the 4-AP.Results are the means ± SEM (n = 5 rats/group).